Methods of detecting micrometastasis of prostate cancer

ABSTRACT

A method of diagnosing prostate micrometastasis is provided by the present invention whereby nucleic acids from a tissue sample from a patient are isolated, nucleic acids from the tissue sample specific for prostate cancer are amplified, or a signal generated by hybridization of a probe specific to a prostate cancer specific nucleic acid is amplified; and detection of amplified nucleic acids is indicative of micrometastasis of prostate cancer.

This is a division of application Ser. No. 08/358,782, filed Dec. 15,1994 U.S. Pat. No. 5,674,682, which is a continuation-in-part of Ser.No. 08/294,611 filed Aug. 23, 1994, now U.S. Pat. No. 5,506,106 which isa continuation of Ser. No. 07/973,322 filed Oct. 29, 1992 abandoned, thedisclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention is directed to methods of detecting prostate cancermicrometastasis.

BACKGROUND OF THE INVENTION

Prostate cancer metastasis will claim the lives of over 30,000 Americansthis year. Boring et al., Cancer Statistics 1991, 19. The mode ofdissemination however, remains very poorly understood. An almostdogmatic view of metastasis holds that prostate cancer cells firstspread through the prostatic capsule then into the lymphatics, andeventually hematogenously travel to bone. Byar et al., Cancer 1972, 30,5; Winter, C. C., Surg. Gynecol. Obstet. 1957, 105, 136; Hilaris et al.,Am. J. Roentgenol. 1974, 121, 832; McLaughlin et al., J. Urol. 1976,115, 89; Jacobs, S. C., Urology 1983, 21, 337; Batson, O. V., Ann. Surg.1940, 112, 138; Saitoh et al., Cancer 1984, 54, 3078-3084; Whitmore, W.F., Jr., Cancer 1973, 32, 1104. However, this model has been based onhistopathologic studies which have significant limitations, and inactuality the sequence of metastatic events remain unknown. Solid tumoranimal experiments suggest that only 0.01% of circulating cancer cellseventually create a single metastatic deposit. Fidler et al., Science1982, 217, 998-1001; Liotta et al., Cancer Res. 1974, 34, 997;Schirrmacher, B., Adv. Cancer Res. 1985, 43, 1-32. Ostensibly, a singlebone metastasis from human prostatic adenocarcinoma (PAC) could begenerated by 10,000 circulating cancer cells (2 cells/1 ml blood). Inthe past, detection of such a low concentration of cells has beendifficult or impossible. Recently, however, Wu et al. used keratin-19(K-19) mRNA PCR to detect breast cancer micrometastasis in patient lymphnodes and bone marrow. Wu et al., Lab. Inv. 1990, 62, 109A. Miyomura etal., also reported the detection of minimal residual acute lymphoblasticleukemia by PCR in patients harboring the Philadelphia chromosome.Miyomura et al., Blood 1992, 79, 1366-1370.

A method of detecting the micrometastasis of prostate cancer would begreatly desirable.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods of detecting prostatecancer micrometastasis in a patient are provided comprising obtaining asample comprising nucleic acids from a patient tissue sample; amplifyingnucleic acids specific for prostate cancer or amplifying a signalgenerated by hybridization of a probe specific to a prostate cancerspecific nucleic acid in said sample; and detecting the presence ofamplified nucleic acids or amplified signal wherein the presence ofamplified nucleic acids or amplified signal indicates micrometastasis ofprostate cancer.

The scope of the present invention also includes a method of detectingcells which express prostate cancer specific sequences comprisingobtaining a sample suspected of having prostate cancer specificsequences comprising nucleic acids; and detecting the presence ofnucleic acids specific for prostate cancer or a signal specific forprostate cancer wherein the presence of nucleic acids or signalindicates prostate cancer.

Prostate cancer specific primer sequences such as and not limited to thesequences GAGGTCCACACACTGAAGTT (SEQ ID NO: 1) GCCTCCTGAAGAATCGATTCCT(SEQ ID NO: 2), GTTGTCTTCCTCACCCTGT (SEQ ID NO: 3); TAAGAAACACTCTGGTCT(SEQ ID NO: 4); AGCCCCAAGCTTACCACCT (SEQ ID NO: 5); CACAATCCGAGACAGGAT(SEQ ID NO: 6); GCCCACTTGTCTGTAATG (SEQ ID NO: 7); CAGGGCACATGGTTCACT(SEQ ID NO: 8), TGGAGTCATCACCTGGcCTGAGGAA (SEQ ID NO: 9), andCCCAACCCTGGCAGGTGTTGTAGC (SEQ ID NO: 10), may be used in theamplification procedure of the present invention. Isolated nucleic acidsequences of SEQ ID NOS: 1-10 are within the scope of the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C shows an agarose gel in which micrometastasis is indicatedby the presence of a 214 base pair (bp) band.

FIG. 2 displays the sequence of a 992 base pair prostate specificantigen (PSA) cDNA PCR fragment, SEQ ID NO: 13, obtained from prostatetissue. PSA exons are boxed. The shaded boxed sequence corresponds tothe postulated binding site to macroglobulins. Oligonucleotide primersequences are underlined. Variations observed in different PSA sequencesin the EMBL data base are indicated in comparison with the sequence cDNAPCR fragment. Numbers in parenthesis in FIG. 2 refer to the sequences inthe following references for the examined sequences: (1) Lundwall andLilja, FEBS Lett., 1987, 214, 317-322; (2) Digby, M. R., et al., NucleicAcids Res., 1989, 17, 2137; (3) Stucka, R. et al., Nucleic Acids Res.,1988, 16, 6226; and (4) Klobeck, H. G., et al., Nucleic Acids Res.,1989, 17, 3981, which were compared to the sequences of the presentinvention.

FIG. 3 is an ethidium bromide stained agarose gel of the PSA RT-PCRproducts from normal prostate tissue (lane 1), benign prostatichypertrophy (BPH) (lanes 2 and 4), prostate adenocarcinoma (lane 3), andmetastatic prostate cancer to a lymph node (lane 5). Lane 6 is anegative control. PCR was performed with PSA specific primers 4 (SEQ IDNO: 4) and 5 (SEQ ID NO: 5).

FIG. 4 displays a possible pattern of prostate cancer metastasis.

FIGS. 5A and 5B is the sequence of PSA-20, the cDNA probe used in thedetection of the amplified product in the methods of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, methods of detecting prostatecancer micrometastasis in a patient are provided comprising obtaining asample comprising nucleic acids from a patient tissue sample; amplifyingnucleic acids specific for prostate cancer or amplifying a signalgenerated by hybridization of a probe specific to a prostate cancerspecific nucleic acid in said sample; and detecting the presence orabsence of amplified nucleic acids or amplified signal wherein thepresence of amplified nucleic acids or amplified signal indicatesmicrometastasis of prostate cancer.

The scope of the present invention also includes a method of detectingcells which express prostate cancer specific sequences comprisingobtaining a sample suspected of having prostate cancer specificsequences comprising nucleic acids; and detecting the presence ofnucleic acids specific for prostate cancer or a signal specific forprostate cancer wherein the presence of nucleic acids or signalindicates prostate cancer.

The method of detecting cells which express prostate cancer specificsequences comprises amplifying nucleic acids specific for prostatecancer or amplifying a signal generated by hybridization of a probespecific to a prostate cancer specific nucleic acid in said sample; anddetecting the presence of the amplified nucleic acids or the amplifiedsignal wherein the presence of amplified nucleic acids or amplifiedsignal indicates micrometastasis of prostate cancer.

A patient suspected of prostate cancer micrometastasis includes apatient diagnosed with prostate cancer who may or may not haveexperienced symptoms associated with metastasis and who has not beenable to be diagnosed by other available methods, such as bone scan andimaging studies, as having micrometastatic or metastatic prostatecancer. In accordance with methods of the present invention, methods ofdetecting micrometastasis of prostate cancer in a patient are providedcomprising obtaining a patient tissue sample for testing. The tissuesample may be solid or liquid, a body fluid sample such as and notlimited to saliva, sputum, mucus, bone marrow, serum, blood, urine,lymph, tears, semen, or feces from a patient suspected of havingprostate cancer. In addition, a tissue sample such as a malignant orbenign tumor, prostate tumor for example, or a may be provided for thedetection of prostate cancer micrometastasis in accordance with thepresent invention.

Nucleic acids, such as DNA (including cDNA) and RNA (including mRNA),are obtained from the patient sample. Preferably RNA is obtained from ablood sample, such as and not limited to a peripheral venous bloodsample. A whole blood gradient may be performed to isolate nucleatedcells and total RNA is extracted such as by the RNazole B method(Tel-Test Inc., Friendswood, Tex.) or by modification of any methodknown in the art such as described in Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989), incorporated herein by reference in its entirety.

Nucleic acid extraction is followed by amplification of the same by anytechnique known in the art. The amplification step incudes the use of atleast one primer sequence which is complementary to a portion of aprostate cancer specific sequence. Prostate cancer specific sequencesare defined for purposes of the present invention to include (and arenot limited to) prostate specific antigen (PSA), prostate specificmembrane antigen (PSM), prostatic acid phosphotase (PAP), and keratin-19sequences.

Sequences useful in the amplification methods include and are notlimited to GAGGTCCACACACTGAAGTT (SEQ ID NO: 1) andGCCTCCTGAAGAATCGATTCCT (SEQ ID NO: 2), GTTGTCTTCCTCACCCTGT (SEQ ID NO:3); TAAGAAACACTCTGGTCT (SEQ ID NO: 4); AGCCCCAAGCTTACCACCT (SEQ ID NO:5); CACAATCCGAGACAGGAT (SEQ ID NO: 6); GCCCACTTGTCTGTAATG (SEQ ID NO:7); CAGGGCACATGGTTCACT (SEQ ID NO: 8), TGGAGTCATCACCTGGCCTGAGGAA (SEQ IDNO: 9), and CCCAACCCTGGCAGGTGTTGTAGC (SEQ ID NO: 10). A Gene Bankversion-70 (Mountain View, Calif.) search confirmed the specificity ofthese primers to PSA and not to the human glandular kallikrein (HMGK)gene which has high homology to the PSA gene. Henttu et al, Biochem.Biophys. Res. Comm. 1989, 160, 903-910, incorporated herein by referencein its entirety. PSA2 (SEQ ID NO: 1) and PSA3 (SEQ ID NO: 2) bindsequences that span intron III of the PSA gene such that PCRamplification yields a 360 bp DNA and a 214 bp RNA product, therebyeliminating the possibility of false positives from DNA contamination.Oligonucleotide primers may be prepared by any method known in the artsuch as by standard phosphoramidite chemistry. (See Sambrook et al.,supra).

Amplification may use oligonucleotides which are complementary toprostate specific antigen gene which do not hybridize to human glandularkallikrein gene. Where a template dependent process of amplificationuses a pair of primers, one primer of the pair may compriseoligonucleotides which are complementary to nucleic acid sequences whichencode prostate cancer specific proteins. The one primer of the pair maybe selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6,7, 8, 9, and 10.

Alternatively, each of the two oligonucleotides in the primer pair maybe specific to a nucleic acid sequence which encodes a prostate cancerspecific protein. The primers may be designed to be complementary toseparate regions of the prostate specific antigen (PSA) gene, forexample. Henttu et al., Biochem. Biophys. Res. Comm. 1989, 160, 903-910,incorporated herein by reference in its entirety. By separate regions ismeant that a first primer is complementary to a 3' region of the PSAgene and a second primer is complementary to a 5' region of the PSAgene. Preferably, the primers are complementary to distinct, separateregions and are not complementary to each other. The primers of SEQ IDNOS: 1-10 are merely exemplary of the primers which may be useful in thepresent invention.

When an amplification method includes the use of two primers, such asthe polymerase chain reaction, the first primer may be selected from thegroup consisting of SEQUENCE ID NOS: 2, 3, 5, 7, and 10, and the secondprimer may be selected from the group consisting of SEQUENCE ID NOS: 1,4, 6, 8, and 9. Any primer pairs which transcribe nucleic acids towardeach other and which are specific for prostate cancer may be used inaccordance with the methods of the present invention.

Total extraction of RNA is preferably carried out. As used herein, theterm "amplification" refers to template-dependent processes andvector-mediated propagation which result in an increase in theconcentration of a specific nucleic acid molecule relative to itsinitial concentration, or in an increase in the concentration of adetectable signal. As used herein, the term template-dependent processis intended to refer to a process that involves the template-dependentextension of a primer molecule. The term template dependent processrefers to nucleic acid synthesis of an RNA or a DNA molecule wherein thesequence of the newly synthesized strand of nucleic acid is dictated bythe well-known rules of complementary base pairing (see, for example,Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A.Benjamin, Inc., Menlo Park, Calif. (1987) incorporated herein byreference in its entirety). Typically, vector mediated methodologiesinvolve the introduction of the nucleic acid fragment into a DNA or RNAvector, the clonal amplification of the vector, and the recovery of theamplified nucleic acid fragment. Examples of such methodologies areprovided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al.,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory,1982, each incorporated herein by reference in its entirety.

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, and in Innis et al., PCR Protocols, Academic Press, Inc., SanDiego Calif., 1990, each of which is incorporated herein by reference inits entirety. Briefly, in PCR, two primer sequences are prepared whichare complementary to regions on opposite complementary strands of thetarget sequence. An excess of deoxynucleoside triphosphates are added toa reaction mixture along with a DNA polymerase (e.g., Taq polymerase).If the target sequence is present in a sample, the primers will bind tothe target and the polymerase will cause the primers to be extendedalong the target sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the target to form reaction products, excessprimers will bind to the target and to the reaction products and theprocess is repeated. Preferably a reverse transcriptase PCRamplification procedure may be performed in order to quantify the amountof mRNA amplified. Polymerase chain reaction methodologies are wellknown in the art.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in EPA No. 320,308, incorporated herein byreference in its entirety. In LCR, two complementary probe pairs areprepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as "target sequences" forligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporatedherein by reference in its entirety, describes an alternative method ofamplification similar to LCR for binding probe pairs to a targetsequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference in its entirety, may also be used asstill another amplification method in the present invention. In thismethod, a replicative sequence of RNA which has a region complementaryto that of a target is added to a sample in the presence of an RNApolymerase. The polymerase will copy the replicative sequence which canthen be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculeshat contain nucleotide 5'- alpha-thio!triphosphates in one strand of arestriction site (Walker, G. T., et al., Proc. Natl. Acad, Sci. (U.S.A.)1992, 89:392-396, incorporated herein by reference in its entirety), mayalso be useful in the amplification of nucleic acids in the presentinvention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e. nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Prostate specific sequences can also be detected using a cyclic probereaction (CPR). In CPR, a probe having a 3' and 5' sequences ofnon-prostate specific DNA and middle sequence of prostate specific RNAis hybridized to DNA which is present in a sample. Upon hybridization,the reaction is treated with RNaseH, and the products of the probeidentified as distinctive products generating a signal which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to a prostatecancer specific expressed nucleic acid.

Still other amplification methods described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,"modified" primers are used in a PCR like, template and enzyme dependentsynthesis. The primers may be modified by labelling with a capturemoiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In thelatter application, an excess of labelled probes are added to a sample.In the presence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labelled probe signals thepresence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh D., et al., Proc. Natl. Acad. Sci.(U.S.A.) 1989, 86:1173, Gingeras T. R., et al., PCT Application WO88/1D315, incorporated herein by reference in their entirety), includingnucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, thenucleic acids can be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a clinical sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has prostate specificsequences. Following polymerization, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second prostate specific primer, followed by polymerization.The double stranded DNA molecules are then multiply transcribed by apolymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAsare reverse transcribed into double stranded DNA, and transcribed onceagainst with a polymerase such as T7 or SP6. The resulting products,whether truncated or complete, indicate prostate cancer specificsequences.

Davey, C., et al., European Patent Application Publication No. 329,822,incorporated herein by reference in its entirety, disclose a nucleicacid amplification process involving cyclically synthesizingsingle-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA),which may be used in accordance with the present invention. The ssRNA isa first template for a first primer oligonucleotide, which is elongatedby reverse transcriptase (RNA-dependent DNA polymerase). The RNA is thenremoved from resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).The resultant ssDNA is a second template for a second primer, which alsoincludes the sequences of an RNA polymerase promoter (exemplified by T7RNA polymerase) 5' to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large "Klenow" fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA("dsDNA") molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller, H. I., et al., PCT Application WO 89/06700, incorporated hereinby reference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA ("ssDNA") followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include "race" disclosed byFrohman, M. A., In: PCR Protocols: A Guide to Methods and Applications1990, Academic Press, New York) and "one-sided PCR" (Ohara, O., et al.,Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:5673-5677), all referencesherein incorporated by reference in their entirety.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting"di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu, D.Y. et al., Genomics 1989, 4:560, incorporated herein by reference in itsentirety), may also be used in the amplification step of the presentinvention.

Following amplification, the presence or absence of the amplificationproduct may be detected. The amplified product may be sequenced by anymethod known in the art, including and not limited to the Maxam andGilbert method, see Sambrook, supra. The sequenced amplified product isthen compared to a sequence known to be in a prostate cancer specificsequence. Alternatively, the nucleic acids may be fragmented intovarying sizes of discrete fragments. For example, DNA fragments may beseparated according to molecular weight by methods such as and notlimited to electrophoresis through an agarose gel matrix. The gels arethen analyzed by Southern hybridization. Briefly, DNA in the gel istransferred to a hybridization substrate or matrix such as and notlimited to a nitrocellulose sheet and a nylon membrane. A labelled probeis applied to the matrix under selected hybridization conditions so asto hybridize with complementary DNA localized on the matrix. The probemay be of a length capable of forming a stable duplex. The probe mayhave a size range of about 200 to about 10,000 nucleotides in length,preferably about 200 nucleotides in length, and more preferably about1462 nucleotides in length. The probe may have the sequence set forth inFIGS. 5A and 5B (SEQ ID NO: 14), or a sequence similar to that set forthin FIGS. 5A and 5B, for example. Mismatches which permit substantialsimilarity to SEQ ID NO: 14, such as and not limited to sequences withsimilar hydrophobicity and hydrophilicity, will be known to those ofskill in the art once armed with the present disclosure. Various labelsfor visualization or detection are known to those of skill in the art,such as and not limited to fluorescent staining, ethidium bromidestaining for example, avidin/biotin, radioactive labeling such as ³² Plabeling, and the like. Preferably, the product, such as the PCRproduct, may be run on an agarose gel and visualized using a stain suchas ethidium bromide. See Sambrook et al., supra. The matrix may then beanalyzed by autoradiography to locate particular fragments whichhybridize to the probe.

A diagnostic kit for detecting micrometastasis of prostate cancercomprising in one or more containers at least one primer which iscomplementary to a prostate cancer specific sequence and a means forvisualizing amplified DNA is also within the scope of the presentinvention. Alternatively, the kit may comprise two primers. In eithercase, the primers may be selected from the group consisting of SEQ IDNOS: 1-10, for example. The diagnostic kit may comprise a pair ofprimers wherein one primer within said pair is complementary to a regionof the prostate specific antigen gene, wherein one of said pair ofprimers is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, a probe specificto the amplified product, and a means for visualizing amplified DNA, andoptionally including one or more size markers, and positive and negativecontrols. The diagnostic kit of the present invention may comprise oneor more of a fluorescent dye such as ethidium bromide stain, ³² P, andbiotin, as a means for visualizing or detecting amplified DNA.Optionally the kit may include one or more size markers, positive andnegative controls, and/or a probe specific to the amplified product.

The present invention is also directed to isolated nucleic acidsequences including and are not limited to the nucleic acid sequencesGAGGTCCACACACTGAAGTT (SEQ ID NO: 1) GCCTCCTGAAGAATCGATTCCT (SEQ ID NO:2), GTTGTCTTCCTCACCCTGT (SEQ ID NO: 3); TAAGAAACACTCTGGTCT (SEQ ID NO:4); AGCCCCAAGCTTACCACCT (SEQ ID NO: 5); CACAATCCGAGACAGGAT (SEQ ID NO:6); GCCCACTTGTCTGTAATG (SEQ ID NO: 7); CAGGGCACATGGTTCACT (SEQ ID NO:8), TGGAGTCATCACCTGGCCTGAGGAA (SEQ ID NO: 9), andCCCAACCCTGGCAGGTGTTGTAGC (SEQ ID NO: 10). Contemplated by thisdefinition are oligonucleotides of about 10 to about 20 codons withinthe coding sequence for prostate specific antigen gene. In addition,mismatches within the sequences identified above, which achieve themethods of the invention, such that the mismatched sequences aresubstantially similar to and thus hybridizable with the sequencesidentified above as SEQ ID NOS: 1-10, are also considered within thescope of the disclosure. Mismatches which permit substantial similarityto any one of SEQ ID NOS: 1-10, such as and not limited to sequenceswith similar hydrophobicity and hydrophilicity, will be known to thoseof skill in the art once armed with the present disclosure. Theoligonucleotides may also be unmodified or modified.

The following examples a:e illustrative but are not meant to be limitingof the invention.

EXAMPLES Example 1

Patient Specimens

Selection of cases was based on the following criteria. Prostate cancerpatients were chosen for analysis if they had: (1) clinically and/orsurgically staged D0-D2 disease (D0=elevated tumor markers with nodemonstrable metastasis, D1=pelvic lymph node involvement,D2=disseminated disease usually to bone) without having received priorhormonal therapy and who had an elevated serum PSA, or (2) stage D3disease (D2 disease that is refractory to hormonal therapy) with anelevated PSA. Negative control patients consisted of female volunteers,and patients with benign prostatic hypertrophy (BPH) proven by biopsy ormen who were on a BPH study protocol. Patients who had surgicalmanipulation of the prostate during the previous year were excluded fromthe study. Positive controls included a lymph node from a patient withknown metastatic PAC tissue from pathologically proven BPH and cDNA PSAplasmid. Henttu et al, Biochem. Biophys. Res. Comm. 1989, 160, 903-910.The protocol was approved by the Internal Review Board of ThomasJefferson University Hospital and written consent was obtained. LNCAPand PC3 human cell lines were obtained from The American Type CultureCollection, (Rockville, Md.).

Example 2

Blood Preparation for RNA Extraction

Approximately six ml of venous blood were obtained with a standardvenipuncture technique using heparinized tubes. Whole blood was mixedwith an equal volume of phosphate buffered saline (PBS) which was thenlayered over eight ml of FICOLL(TM) (Pharmacia Uppsala, Sweden) in a 15ml polystyrene tube. The gradient was centrifuged at 200 g for 30minutes at 5° C. The lymphocyte and granulocyte layer (approximately 5ml) was carefully aspirated and re-diluted up to 50 ml with PBS in a 50ml tube which was then centrifuged at 1800 g for 20 minutes a 5° C.Supernatant was discarded, and the pellet containing nucleated cells wasused for RNA extraction using the RNazole B method, as described by thecompany (Tel-Test Inc., Friendswood, Tex.).

Example 3

Oligonucleotide primers and probes

PSA2 (5-GAGGTCCACACACTGAAGTT, SEQ ID NO: 1) and PSA3(5-CCTCCTGAAGAATCGATTCCT, SEQ ID NO: 2) oligonucleotide primers werecustom designed with high specificity to the PSA gene; a Gene Bankversion-70 (Mountain View, Calif.) search confirmed the specificity ofthese primers to PSA and not to the human glandular kallikrein (HMGK)gene which has 75-85% homology to the PSA gene. Henttu et al, Biochem.Biophys. Res. Comm. 1989, 160, 903-910. The primers were synthesized andgel purified by the City of Hope DNA Synthesis Laboratory (Duarte,Calif.). PSA2 (SEQ ID NO: 1) and PSA3 (SEQ ID NO: 2) bind sequences thatspan intron III such that PCR amplification yielded a 360 bp DNA and a214 bp RNA product. Previously published actin PCR primer sequences wereused to rule out degraded RNA, and amplification with actinoligonucleotide primers A1 TCATCACCATTGGCAATGAG (SEQ ID NO: 11) and A2CACTGTGTTGGCGTACAGGT (SEQ ID NO: 12) yielded a 154 bp RNA and a 250 bpDNA product. Ben-Ezra et al., J. Histochem Cytochem. 1991, 39, 351-354,incorporated herein by reference in its entirety.

Example 4

Reverse Transcriptase Reaction and Polymerase Chain Reaction

The reverse transcriptase reaction and PCR amplification were performedsequentially without interruption in a Perkin Elmer 9600 PCR machine(Emeryville, Calif.). 400 ng of total RNA in 20 μl DEPC(Diethyl-pyrocarbonate)-treated water were placed in a 65° C. water bathfor five minutes then quickly chilled on ice immediately prior to theaddition of PCR reagents. The 50 μl total PCR volume consisted of 2.5units Taq polymerase (Perkin Elmer, Emeryville, Calif.), 2 units AMVreverse transcriptase (Boehringer Mannheim, Indianapolis, Ind.), 200 μMeach of dCTP, DATP, dGTP, and dTTP (Perkin Elmer, Emeryville, Calif.),18 PM each primer, 10 mM Tris-HCL, 50 mM KCl, and 2 mM MgCl₂ (PerkinElmer, Emeryville, Calif.). PCR conditions were as follows: cycle 1 wasperformed at 42° C. for 15 minutes, then 97° C. for 15 seconds (onecycle); cycle 2 was performed at 95° C. for one minute, then 60° C. forone minute and 72° C. for 30 seconds (15 cycles); cycle 3 was performedat 95° C. for one minute, then 60° C. for one minute, and 72 degrees forone minute (10 cycles); cycle 4 was performed at 95° C. for one minute,then 60° C. for one minute and 72° C. for two minutes (8 cycles); cycle5 was 72° C. for 15 minutes (one cycle); and the final cycle was held at4° C. until the sample was taken out of the machine. The 50 μl PCRproducts were concentrated down to 10 μl with vacuum centrifugation andthe entire sample was then run on a thin three percent Tris-borate-EDTA(TBE) agarose gel containing ethidium bromide. All specimens wereanalyzed at least twice to confirm a positive or negative outcome.

The potential risk of false positives from cross contamination wasavoided by performing RT PCR in a single tube without interruption andusing filtered pipet tips. Sensitivity was enhanced by using highamounts of Taq polymerase, progressively increasing extension times, andanalyzing the entire 50 μl PCR product on thin ethidium bromide agarosegels. These measures ensured a high fidelity assay while maintainingtechnical simplicity.

Prostate human tissue specimens, tissue culture cell lines and a PSAcDNA plasmid, cloned and described by Henttu and Vihko; Henttu et al.,Biochem. Biophys. Res. Comm. 1989, 160, 903-910, were used as positivecontrols, and they demonstrated the 214 bp bands as shown in FIG. 1A. Apelvic lymph node with metastatic prostatic adenocarcinoma (PAC), aprimary prostate cancer, and a BPH specimen all produced strong PSA PCRsignals. The LNCAP and PC-3 human prostate cell lines produced weakersignals.

Example 5

Sequencing

Specificity of the primers to the PSA gene was confirmed with DNAsequence analysis of the amplified 214 bp fragment (FIG. 1C) which inthis segment had very little homology to the human glandular kallikrein,HMGK, gene.

The 214 bp product was purified with a Qiagen PCR Product Purificationkit (Qiagen, Chatsworth, Calif.) as described by the manufacturer. Onemicrogram of the PCR product underwent a PCR sequencing reaction byusing the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer9600 PCR Machine, as described by Applied Biosystems (AppliedBiosystems, Foster, Calif.). The sequenced product was purified usingcentri-sep columns (Princeton Separations, Adelphia, N.J.) as describedby the company. This product was then analyzed with an ABI Model 373ADNA sequencing system (Applied Biosystems, Foster, Calif.) integratedwith a Macintosh IIci computer.

Example 6

Detection of Circulating Hematogenous Micrometastasis

Twelve prostate cancer patients and 17 control patients underwent RT PCRanalysis on PSA and actin RNA extracted from blood, as described inExamples 1 through 4. The results are reported in Table 1. All casesdemonstrated satisfactory RNA quality by actin PCR (FIGS. 1A-1C, bottomrow). Of the 12 human prostatic adenocarcinoma (PAC) patients withmetastatic disease, four cases (33%) had positive PSA signals indicatingthe presence of prostatic epithelial cells in the peripheral venousblood. These four cases consisted of two stage D1 patients, one stage D2patient, and one stage D3 patient (N=1) (FIG. 1A, top row). The 17negative controls, which consisted of eight volunteer women and nine menwith BPH, all had undetectable PSA mRNA by RT PCR. These data indicatethat RT PCR of the PSA RNA gene can be used to specifically detectcirculating hematogenous micrometastasis in patients with stage D1-D3pathology. These findings are in agreement with studies by Hamby et al.who detected circulating PSA positive cells in patients with metastaticprostate cancer by flow cytology and immunohistology. Hamby et al., Br.J. Urol. 1992, 69, 392-396, incorporated herein by reference in itsentirety.

Micrometastasis was not detected in eight of twelve prostate cancerpatients consisting of two stage D3 patients, two stage D1 patients, andfour stage D0 patients. Results indicate that the prostate cancer cellsmay be more concentrated in the "buffy coat" of the FICOLL(TM) gradient.The PSA signal was stronger in the RNA extracted from cells obtainedonly from the "buffy coat" (FIG. 1B, lane 8) compared to those isolatedfrom the entire FICOLL(TM) layer (FIG. 1B, lane 7) in the same prostatecancer patient. These findings are in agreement with those of Harty etal. who found that prostatic epithelial cells migrate into the "buffycoat". Harty et al., J. Surg. Res. 1979, 26, 411-416, incorporatedherein by reference in its entirety. In order to enhance the detectionof micrometastasis, analysis may thus focus on these buffy coat cells.

                  TABLE 1    ______________________________________    HEMATOGENOUS MICROMETASTASIS SUMMARY    Prostate Cancer Patients                     Control Patients          No. of   Positive         No. of Positive    Stage Patients PSA/PCR   Source Patients                                           PSA/PCR    ______________________________________    D0    4        0         Females                                    8      0                             BPH    D1    4        2                9      0    D2    1        1    D3    3        1    Total 12       4 (33%)          17     0    ______________________________________

These data support animal experimental work suggesting that only thefittest tumor cells survive the metastatic cascade. A large number ofcirculating cancer cells (greater than 10,000 cells in a 70 kg human)may be required in order for a small percentage (less than 0.01%) ofcells to survive and create a metastatic deposit. The finding of cancercells in the peripheral venous blood suggests that these cells circulatethrough all organ capillary beds while forming metastatic coloniesalmost exclusively in bone. These data lend credence to the theory thatthe distribution of prostate cancer metastasis is determined by organtropism, and not strictly anatomic factors such as Batson's vertebralvenous plexus. Organ tropism may be determined by (a) preferentialgrowth of cells in certain organs, (b) preferential adherence of cellsto certain endothelial strictures, and (c) chemotaxis. Finally, thediscovery of prostate cancer cells in circulation of patients with D1pathology calls into question the hypothesis that blood metastasis is aterminal event in the natural history of prostate cancer metastasis. Tothe contrary, these data point to the possibility that prostate cancercells may spread to the neurovascular structures and then into the bloodcirculation, and lymphatic spread may be an associated event that is notan essential step for dissemination, FIG. 4.

Example 7

Patient Specimens

Analysis was performed on five prostate specimens from three patients.Histologically confirmed normal, benign prostatic hypertrophy (BPH), andadenocarcinoma tissue samples were obtained all within the same glandfrom a single patient undergoing radical prostatectomy for localizedprostate cancer. The BPH tissue was also procured after transurethralprostatectomy in a second patient with a serum PSA of 4 ng/ml. Ametastatic prostate cancer specimen was obtained from a grossly diseasediliac node taken during a laparoscopic pelvic lymph node dissection in athird patient with stage D1 disease and a serum PSA of 27 ng/ml.Immediately after removal, tissues were stored at -70° C. prior to RNAextraction.

RNA Extraction

Total RNA extraction was performed using the RNazol B method asdescribed by the manufacturer (Tel-Test, Inc., Friendswood, Tex.).Tissue samples were lysed in 3 ml RNAzol B and a homogenizer was appliedto facilitate lysis when necessary. After addition of 0.3 ml ofchloroform, the samples were vortexed and centrifuged at 12,000 g for 15minutes at 4° C. The upper phase, containing total cellular RNA, wascarefully removed and precipitated for 15 minutes with one volume ofisopropyl alcohol. The pellet was rinsed twice with cold 75% ethanol,dried briefly in a speedvac apparatus and dissolved in 50-200 μl ofwater.

cDNA Synthesis

Total RNA (2 mg) was used for the synthesis of the first strand ofcomplementary DNA (cDNA) using the SuperScript II reverse transcriptase(GIBCO-BRL). Total RNA from all specimens was isolated and reversetranscribed to cDNA using a primer having the sequence of SEQ ID NO: 4,specific for the PSA 3' untranslated region (nucleotides 992-972).Briefly, RNA and 20 pM of PSA primer 4 (SEQ ID NO: 4) were firstdenatured for 5 minutes at 70° C., chilled on ice for one minute andthen incubated for one hour at 42° C. in 20 ml of a reaction mixturecontaining 1× first strand buffer, 250 mM/L dTNPs, 10 mM DTT and 200U ofSuperScript II reverse trancriptase.

Oligonucleotide Primers

Oligonucleotide sequences, GAGGTCCACACACTGAAGTT (SEQ ID NO: 1)GCCTCCTGAAGAATCGATTCCT (SEQ ID NO: 2), GTTGTCTTCCTCACCCTGT (SEQ ID NO:3); TAAGAAACACTCTGGTCT (SEQ ID NO: 4); AGCCCCAAGCTTACCACCT (SEQ ID NO:5); CACAATCCGAGACAGGAT (SEQ ID NO: 6); GCCCACTTGTCTGTAATG (SEQ ID NO:7); CAGGGCACATGGTTCACT (SEQ ID NO: 8), TGGAGTCATCACCTGGCCTGAGGAA (SEQ IDNO: 9) and CCCAACCCTGGCAGGTGTTGTAGC (SEQ ID NO: 10); were chosen withinPSA gene regions which maximize mismatches with other genes of the samefamily. SEQ ID NO: 2 has also been used with the first "G" deleted, suchthat the primer is also useful as a 21mer. The location of the primersis set forth in FIG. 2.

Polymerase Chain Reaction Procedure

One fifth of the cDNA preparation was amplified in 40 μl of PCR mixcontaining 1× PCR buffer (Boehringer Mannheim), 10 pM of each primer,250 mM/L of dNTPs and 1.25 units of Taq DNA polymerase (BoehringerMannheim). Each PCR cycle included denaturation at 94° C. for one minute(2 minutes for the first cycle), annealing at 56° C. for one minute andextension at 72° C. for two minutes (five minutes for the lastextension). cDNA fragments were amplified using the same 3' primer ofSEQ ID NO: 4 (PSA primer 4) with a primer specific for the first 20nucleotides of the PSA cDNA SEQ ID NO: 5 (PSA primer 5). Thirty cycleswere carried out using the PSA primers 5 and 4. PCR products werevisualized in ethidium bromide stained 1.4% agarose gels and thenpurified using a gel extraction kit (Qiagen) following themanufacturer's instructions. The 992 bp PSA cDNA fragments,corresponding to the entire translated and part of the 3' untranslatedregion, were purified from preparative agarose gel electrophoresis andthe DNA sequence was determined. This process was repeated with variouscombinations of primers set forth in FIG. 2, including primercombinations of SEQ ID NOS: 5 and 6, SEQ ID NOS: 1 and 6, SEQ ID NOS: 3and 2, SEQ ID NOS: 7 and 8, SEQ ID NOS: 8 and 3, SEQ ID NOS: 8 and 1,SEQ ID NOS: 8 and 5, SEQ ID NOS: 4 and 7, SEQ ID NOS: 4 and 3, SEQ IDNOS: 4 and 1, and SEQ ID NOS: 4 and 5. Any of the primers withunderlined arrows pointing toward each other (FIG. 2), which transcribetoward each other such that each strand is transcribed in the 3'direction, may be used together.

Sequencing

An automated 373A DNA sequencer (Applied Biosystems) and dye terminatorkits from the same manufacturer were used for direct sequencing of thePSA cDNA fragments by the dideoxynucleotide chain termination methodusing fluorescent labels. The coding and non-coding strands of eachfragment were sequenced with primers generating overlapping sequencedata. The cDNA fragment was sequenced at least three times in both the5' and 3' directions. Purity of cancer was ensured by analyzing ametastatic lymph node which would express PSA derived only from highlymalignant prostatic cells.

Sequences were reassembled and analyzed using the SAP program. Thenormal, BPH, and prostate cancer cDNA sequence data of both strands werealigned and a computer analysis revealed 100% match with no evidence ofmutation in prostate cancer as compared to normal tissue. Sequences werethen compared to the published PSA cDNA sequences. The gene bank searchdemonstrated the following variations with the published PSA sequences:a dinucleotide (GT) deletion at position 38-39 when compared to Lundwalland Lilja's sequence (gene bank accession number x05332); a T to C atposition 91 and an A to G at position 115 when compared to sequencenumber x13941, a G to A at position 169 and a G to A at position 843-844after comparison with sequence number x14810. The perfect identitybetween our sequences obtained from 5 specimens in three differentpatients, suggests that the variations observed in the publishedsequences may be due to genetic polymorphisms or technical artifacts.

The complete coding sequence and part of the 3' untranslated PSA mRNAgene sequence isolated from BPH, normal, and malignant prostate tissuespecimens were obtained and aligned. Computer analysis demonstrated anidentical PSA cDNA match between the PSA expressed by BPH and normaltissue versus prostate cancer. The data indicates that a single geneticform of PSA is expressed in benign and malignant prostate tissue. It ispossible that a mutated PSA gene might be specifically expressed incells with bone invasive capabilities. Alternatively, the occurrence ofaltered PSA complexing and glycosylation in prostate cancer patients maybe a result of post-translational events.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 14    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    # 20               AGTT    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #                 22TTC CT    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    # 19               TGT    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #  18              CT    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    # 19               CCT    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #  18              AT    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #  18              TG    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #  18              CT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #               25 CCTG AGGAA    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #                24GTTG TAGC    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    # 20               TGAG    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    # 20               AGGT    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 992 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - AGCCCCAAGC TTACCACCTG CACCCGGAGA GCTGTGTNNC ACCATGTGGG TC - #CCGGTTGT      60    - CTTCCTCACC CTGTCCGTGA CGTGGATTGG TGCTGCACCC CTCATCCTGT CT - #CGGATTGT     120    - GGGAGGCTGG GAGTGCGAGA AGCATTCCCA ACCCTGGCAG GTGCTTGTAG CC - #TCTCGTGG     180    - CAGGGCAGTC TGCGGCGGTG TTCTGGTGCA CCCCCAGTGG GTCCTCACAG CT - #GCCCACTG     240    - CATCAGGAAC AAAAGCGTGA TCTTGCTGGG TCGGCACAGC CTGTTTCATC CT - #GAAGACAC     300    - AGGCCAGGTA TTTCAGGTCA GCCACAGCTT CCCACACCCG CTCTACGATA TG - #AGCCTCCT     360    - GAAGAATCGA TTCCTCAGGC CAGGTGATGA CTCCAGCCAC GACCTCATGC TG - #CTCCGCCT     420    - GTCAGAGCCT GCCGAGCTCA CGGATGCTGT GAAGGTCATG GACCTGCCCA CC - #CAGGAGCC     480    - AGCACTGGGG ACCACCTGCT ACGCCTCAGG CTGGGGCAGC ATTGAACCAG AG - #GAGTTCTT     540    - GACCCCAAAG AAACTTCAGT GTGTGGACCT CCATGTTATT TCCAATGACG TG - #TGTGCGCA     600    - AGTTCACCCT CAGAAGGTGA CCAAGTTCAT GCTGTGTGCT GGACGCTGGA CA - #GGGGGCAA     660    - AAGCACCTGC TCGGGTGATT CTGGGGGCCC ACTTGTCTGT AATGGTGTGC TT - #CAAGGTAT     720    - CACGTCATGG GGCAGTGAAC CATGTGCCCT GCCCGAAAGG CCTTCCCTGT AC - #ACCAAGGT     780    - GGTGCATTAC CGGAAGTGGA TCAAGGACAC CATCGTGGCC AACCCCTGAG CA - #CCCCTATC     840    - AACCCCCTAT TGTAGTAAAC TTGGAACCTT GGAAATGACC AGGCCAAGAC TC - #AAGCCTCC     900    - CCAGTTCTAC TGACCTTTGT CCTTAGGTGT GAGGTCCAGG GTTGCTAGGA AA - #AGAAATCA     960    #         992      ACCA GAGTGTTTCT TA    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1462 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    - ATTCCGCCGG AGAGCTGTGT CACGATGTGG GTCCCGGTTG TCTTCCTCAC CC - #TGTCCGTG      60    - ACGTGGATTG GTGCTGCACC CCTCATCCTG TCTCGGATTG TGGGAGGCTG GG - #AGTGCGAG     120    - AAGCATTCCC AACCCTGGCA GGTGCTTGTG GCCTCTCGTG GCAGGGCAGT CT - #GCGGCGGT     180    - GTTCTGGTGC ACCCCCAGTG GGTCCTCACA GCTGCCCACT GCATCAGGAA CA - #AAAGCGTG     240    - ATCTTGCTGG GTCGGCACAG CCTGTTTCAT CCTGAAGACA CAGGCCAGGT AT - #TTCAGGTC     300    - AGCCACAGCT TCCCACACCC GCTCTACGAT ATGAGCCTCC TGAAGAATCG AT - #TCCTCAGG     360    - CCAGGTGATG ACTCCAGCCA CGACCTCATG CTGCTCCGCC TGTCAGAGCC TG - #CCGAGCTC     420    - ACGGATGCTG TGAAGGTCAT GGACCTGCCC ACCCAGGAGC CAGCACTGGG GA - #CCACCTGC     480    - TACGCCTCAG GCTGGGGCAG CATTGAACCA GAGGAGTTCT TGACCCCAAA GA - #AACTTCAG     540    - TGTGTGGACC TCCATGTTAT TTCCAATGAC GTGTGTGCGC AAGTTCACCC TC - #AGAAGGTG     600    - ACCAAGTTCA TGCTGTGTGC TGGACGCTGG ACAGGGGGCA AAAGCACCTG CT - #CGGGTGAT     660    - TCTGGGGGCC CACTTGTCTG TAATGGTGTG CTTCAAGGTA TCACGTCATG GG - #GCAGTGAA     720    - CCATGTGCCC TGCCCGAAAG GCCTTCCCTG TACACCAAGG TGGTGCATTA CC - #GGAAGTGG     780    - ATCAAGGACA CCATCGTGGC CAACCCCTGA GCACCCCTAT CAACTCCCTA TT - #GTAGTAAA     840    - CTTGGAACCT TGGAAATGAC CAGGCCAAGA CTCAAGCCTC CCCAGTTCTA CT - #GACCTTTG     900    - TCCTTAGGTG TGAGGTCCAG GGTTGCTAGG AAAAGAAATC AGCAGACACA GG - #TGTAGACC     960    - AGAGTGTTTC TTAAATGGTG TAATTTTGTC CTCTCTGTGT CCTGGGGAAT AC - #TGGCCATG    1020    - CCTGGAGACA TATCACTCAA TTTCTCTGAG GACACAGATA GGATGGGGTG TC - #TGTGTTAT    1080    - TTGTGGGGTA CAGAGATGAA AGAGGGGTGG GTACCACACT GAGAGAGTGG AG - #AGTGACAT    1140    - GTGCTGGACA CTGTCCATGA AGCACTGAGC AGAAGCTGGA GGCACAACGC AC - #CAGACACT    1200    - CACAGCAAGG ATGGAGCTGA AAACATAACC CACTCTGTCC TGGAGGCACT GG - #GAAGCCTA    1260    - GAGAAGGCTG TGAGCCAAGG AGGGAGGGTC TTCCTTTGGC ATGGGATGGG GA - #TGAAGTAA    1320    - GGAGAGGGAC TGGACCCCCT GGAAGCTGAT TCACTATGGG GGGAGGTGTA TT - #GAAGTCCT    1380    - CCAGACAACC CTCAGATTTG ATGATTTCCT AGTAGAACTC ACAGAAATAA AG - #AGCTGTTA    1440    #               1462AAT CC    __________________________________________________________________________

What is claimed is:
 1. A method of detecting prostate cancermicrometastasis in a patient having stage D1, D2, or D3 prostate cancercomprising:obtaining a sample comprising nucleic acids from a patienttissue sample; amplifying nucleic acids specific for prostate cancer oramplifying a signal generated by hybridization of a probe specific to aprostate cancer specific nucleic acid in said sample, comprisinghybridization to at least one prostate cancer specific primer which iscomplementary to a prostate cancer specific gene which does nothybridize to human glandular kalllikrein gene; and detecting thepresence of the amplified nucleic acids or the amplified signal whereinthe presence of amplified nucleic acids or amplified signal indicatesmicrometastasis of prostate cancer.
 2. The method of claim 1 wherein thenucleic acids specific for prostate cancer comprise nucleic acidsencoding proteins selected from the group consisting of prostatespecific antigen, prostate specific membrane antigen, prostatic acidphophatase.
 3. The method of claim 1 wherein said primer is one primerof a pair of primers.
 4. The method of claim 1 wherein saidamplification step comprises performing a procedure selected from thegroup consisting of polymerase chain reaction, ligase chain reaction,repair chain reaction, cyclic probe reaction, nucleic acid sequencebased amplification, strand displacement amplification, and Qβreplicase.
 5. The method of claim 1 wherein said amplification stepcomprises performing a polymerase chain reaction, wherein saidpolymerase chain reaction comprises a first primer and a second primer,wherein said first primer is selected from the group consisting ofSEQUENCE ID NOS: 2, 3, 5, 7, and 10, and said second primer is selectedfrom the group consisting of SEQUENCE ID NOS: 1, 4, 6, 8, and
 9. 6. Themethod of claim 1 wherein said amplification step comprises performing apolymerase chain reaction wherein said polymerase chain reactioncomprises a first primer and a second primer, wherein said first primeris SEQ ID NO: 9, and said second primer is SEQ ID NO:
 10. 7. The methodof claim 1 wherein said amplification step comprises performing apolymerase chain reaction, wherein said polymerase chain reactioncomprises a pair of primers, wherein one primer of said pair iscomplementary to prostate specific antigen gene and does not hybridizeto human glandular kallikrein gene.
 8. The method of claim 7 wherein theprimer that is complementary to prostate specific antigen gene isselected from the group consisting of SEQUENCE ID NOS: 1, 2, 3, 4, 5, 6,7, 8, 9, and
 10. 9. The method of claim 7 wherein the primer that iscomplementary to prostate specific antigen gene is SEQ ID NO:
 9. 10. Themethod of claim 7 wherein the primer that is complementary to prostatespecific antigen gene is SEQ ID NO:
 10. 11. The method of claim 1wherein said patient tissue sample is selected from the group consistingof saliva, sputum, mucus, bone marrow, serum, blood, urine, lymph,tears, or semen.
 12. The method of claim 1 wherein said nucleic acidscomprise RNA obtained from cells from the buffy coat of the FICOLL(TM)layer of a prepared blood sample gradient.
 13. A method of detectingcells which express prostate cancer specific sequences comprisingobtaining a sample suspected of having prostate cancer specific nucleicacids having stage D1, D2, or D3 prostate cancer; and detecting thepresence of nucleic acids specific for prostate cancer or a signalspecific for prostate cancer wherein the presence of nucleic acids orsignal indicates prostate cancer comprising amplifying nucleic acidsspecific for prostate cancer or amplifying a signal generated byhybridization of a probe specific to a prostate cancer specific nucleicacid in said sample; anddetecting the presence of the amplified nucleicacids or the amplified signal, comprising hybridization to at least oneprostate cancer specific primer which is complementary to a prostatecancer specific gene which does not hybridize to human glandularkallikrein gene, wherein the presence of amplified nucleic acids oramplified signal detects cells which express prostate cancer specificsequences.